Deoxyinosine 3′ Endonuclease, a Novel Deoxyinosine‐Specific Endonuclease from Escherichia coli

Deoxyinosine in DNA arises from deamination of deoxyadenosine or misincorporation of dITP during replication. Deamination can be spontaneous or caused by nitrous acid or ionizing radiation.' Deoxyinosine in DNA is potentially mutagenic, resulting in A to G transitions.* One pathway for the repair of deoxyinosine may involve hypoxanthine DNA glycosylase, an enzyme that has been partially p ~ r i f i e d . ~ . ~ But little is known about the mechanism of its action. In an effort to purify this enzyme, we isolated a novel deoxyinosine-specific endonuclease, deoxyinosine 3' endonuclease. Deoxyinosine 3' endonuclease was purified from E. coli strain BW 434 (nth-xth-). Deoxyinosine-containing bacteriophage PM2 DNA, prepared by nick translation of dITP in place of dGTP, was used as a substrate for detection of deoxyinosine-specific endonuclease activity by the alkaline fluorometric assay of Kowal~ki .~ Cell paste was first homogenized by Braun MSK homogenizer. After removal of nucleic acids by PEG precipitation, the enzyme was purified by chromatography on S Sepharose Fast Flow, followed by FPLC on Mono S, Mono Q, and Phenyl Superose. At the last step of purification, the preparation represented 24,800fold purification over the crude extract, with a 43% yield of activity. Silver-stained SDS-PAGE revealed a major polypeptide of 26 kDa with two minor contaminants. We eluted the 26-kDa polypeptide from SDS-PAGE and renatured and recovered the deoxyinosine-specific activity. Activity gel electrophoresis was also used to confirm that the deoxyinosine-specific endonuclease migrated as a 26-kDa polypeptide. The enzyme has an obligatory requirement for Mg2+ at a concentration of 50 pM. Fifty pM of CoCl, or 10 pM MnCl, could partially replace the requirement of Mgz+. The optimal pH for the enzyme is 7.5. The enzyme was found to be active on oligonucleotides containing I/T, I/C, I/A, and I/G pairs. It is also active on single-stranded DNA containing deoxyinosine, albeit at a lower rate. Deoxyinosine 3' endonuclease was also found to be active on DNA containing abasic site (AP) or urea. It cleaves the strand containing deoxyinosine or the AP site, but not the complementary strand. It does not cleave oligonucleotides with A/T pairs. It was revealed by 3' and 5' end labeling of the oligonucleotide that the enzyme cuts the second phosphodiester bond 3' to deoxyinosine. This was true for all deoxyinosine pairs and deoxyinosine-containing single-stranded DNA, as well as the AP-containing oligonucleotide. Deoxyinosine 3' endonuclease creates a nick with 3' hydroxyl group since the product of the enzyme is a substrate for nick translation. The enzyme is different from hypoxanthine DNA glycosylase and displays a novel AF' endonuclease activity different from those of class I and class I1 AP endonucleases.